Wet lab | iGEM Hamburg 2025

Achievements

We expressed protein constructs for GFP and a nanobody against GFP in BL21(DE3) E. coli and validated the expression through a Western Blot.

Nanobody construct design

Nanobody Description

As nanobodies seemed like the perfect candidate for capturing α-amanitin and protecting cells against poisoning damage (see our Background page), we started the design process of our nanobody constructs by collecting requirements, both therapeutic and in terms of production.

First of all, our nanobody should effectively neutralize alpha-amanitin. Since the toxin binds to the RNA polymerase with very high affinity (KD = 3-4 nM [1] ), our nanobody needs to have a high affinity to the toxin as well to be able to compete with the polymerase. This is generally possible to achieve, although it can be much harder to design nanobodies with high affinities against small targets such as peptides (expert interview de Marco and Schöder).

We also aim for as little side effects as possible, as patients that would be treated with our antidote are already weakened by the toxin and often in critical condition. Fortunately, nanobodies already exist as therapeutics and show no immunological effects [2] , making them an ideal candidate for our application.

Since the RNA polymerase is located in the cell nucleus, we need to deliver our nanobody into the nucleus of affected cells. To achieve this, we added a nuclear localization sequence (NLS) originally derived from the simian virus 40 [3] . This NLS is recognized by transporters that help import respective proteins into the nucleus [4].

Nanobodies are also promising regarding transport and storage. As they are highly stable and do not need to be cooled down to extreme temperatures, they provide a treatment alternative for remote locations or hospitals that lack elaborate cooling options.

To make our antidote affordable, we were looking for an inexpensive way of production that can be upscaled easily. Since nanobodies can be produced recombinantly in procaryotic expression systems, we chose the commonly available E. coli strain BL21(DE3) for production. This way, our antidote production is also animal-free.

We want to be able to produce a pure nanobody that is free of any procaryotic traces. To achieve this, we added two things to our design: first, we want our nanobody to be extracellularly secreted by our E. coli. We added a signal peptide called pelB to the nanobody, which directs the protein into the periplasm of the bacteria. This helps form the disulfide bond our nanobody contains and leads to improved folding. Once in the periplasm, proteins can be further secreted into the medium around the bacteria using an aspartate linker [5] . The extracellular secretion can lead to higher protein production and easier downstream isolation and purification [6] . Secondly, we added tags for affinity purification to our nanobody. As our nanobody is a rather small protein, we chose two small tags, a strep-tag II and a 6x Histidine tag, and created constructs with each of them, respectively.

Finally, we anticipated optimization and engineering steps necessary to further develop our nanobody. We added protease sites for the TEV protease and the HRV 3C protease. This way we are able to cut off the aspartate linker required for secretion and the tag required for purification to minimize our nanobody’s immunogenicity for therapeutic applications. We also included several restriction enzyme recognition sites into our design. Thus, we can cut out the nuclear localization sequence to create a nanobody capturing the toxin in the bloodstream or in the cytosol of the cells. We are also able to switch out the coding sequence for the nanobody, so we can use our constructs to produce nanobodies with different targets, for example against other intracellular toxins.

Important note

Unfortunately, we did not check our first design with iGEM’s parts requirements. We included restriction enzyme recognition sites that are not allowed in the BioBrick RFC[10] system. Additionally, our nanobody construct also includes restriction sites incompatible with the Type IIS RFC[1000] system.

Interactive Plasmid Maps

Our final construct contains a strong RBS (BBa_B0030) for increased protein expression. The reading frame starts with a pelB sequence (BBa_K2114998) for periplasmic secretion, followed by a 5x aspartate linker for subsequent extracellular secretion [5] . We then added a HRV 3C protease cleavage site to be able to remove the 5x aspartate linker. This is followed by the CDS for a nanobody against GFP (BBa_K2114998), which we will use as a placeholder for our own nanobody. Additionally, we designed constructs containing the CDS for GFP as a control for the production of the nanobody. Right behind the CDS, we added a linker and a 3x NLS for nuclear localization [5] . After this, we added a second protease cleavage site, this time for the TEV protease for cutting off the 6x Histidine tag or strep-tag II. We ordered the constructs for GFP and the anti-GFP nanobody in a pET blank vector lacking the RBS from our sponsor, Twist Bioscience, and named our constructs GFP_his and aGFP_his.

Engineering and troubleshooting:

We were not able to identify any expression of our protein constructs using our first design idea. To confirm that our expression conditions and our E. coli strain were working in general, we used a positive expression control. This positive expression control consisted of a protein used in our lab before, which was known to be expressed in BL21 (DE3) E. coli. It is regulated through the lac-operon as well, but has no secretion signals for the periplasm or the extracellular space. We were able to recover this positive control in our SDS-PAGE analyses, indicating that protein production itself was not impacted. So we went back to the drawing board and varied different aspects of our constructs and plasmids.

First redesign

We took a closer look at parts using the RBS from part BBa_B0030 and noticed that they include a shorter distance between the annotated RBS and the start codon of the CDS. While we introduced seven basepairs 3’ of the RBS, part BBa_K1956022 contains six basepairs 3’ of the RBS and was expressed in DH5-alpha E. coli. in the backbone plasmid pSB1A2. Part BBa_K5299200 only contains one basepair 3’ of the RBS and was expressed in BL21(DE3) E. coli. in the backbone plasmid pDGB3a1. We adjusted the distance in our construct to one basepair as well and named the new constructs RBS1_GFP_his and RBS1_aGFP_his. We ordered these constructs cloned into the pET blank vector from Twist Bioscience that does not contain an RBS.

Second redesign

We ordered our constructs inserted into a pET expression vector for E. coli which was available at our sponsor Twist Bioscience. To rule out problems regarding the usage of the RBS (BBa_B0030) in a pET vector, we designed a new version of our construct that contained the RBS and spacing commonly used in the pET vector system. We named these new constructs RBSp_GFP_his and RBSp_aGFP_his. These constructs were cloned into a pET blank vector without an RBS by Twist Bioscience.

Third redesign

To match iGEM’s rules for part compatibility, we re-designed our construct by switching out restriction sites and including only those allowed in both the BioBrick RFC[10] and the Type IIS RFC[1000] system. We also excluded the RBS from the construct design and named the new constructs part_GFP_his and part_aGFP_his. We ordered them cloned into the pET blank vector containing an RBS.

Fourth redesign

Our first construct design includes two protease cleavage sites as well as the linker made of five aspartates in a row. As we were hypothesizing that this linker and the protease sites might be difficult to translate or fold for our E. coli, we excluded them in a fourth redesign. The new constructs only include a pelB, the CDS of the protein, a 3x NLS and a 6x His-Tag and were named part_bas_GFP_his and part_bas_aGFP_his. We ordered them in the pET blank vector from Twist Bioscience, which contains an RBS.

Modification

As we had introduced several tags and signal sequences into our construct, we sought to find out if one of them prevented the production of our proteins. We started by removing the 3x NLS at the C-terminus of the protein using restriction digestion. This way, we wanted to check if the sequence somehow leads to expression issues.

Nanobody construct results

Getting started - plasmid amplification, transformation and validation

Our overall aim in the WetLab was to set up a production pipeline for a recombinant nanobody construct in E. coli. In addition to a construct containing a nanobody against GFP, we also worked with a construct for GFP that served both as a control for construct expression and a future binding partner for the anti-GFP nanobody. We ordered our constructs from Twist Bioscience who synthesized the inserts for us and cloned them into a pET blank vector carrying a kanamycin resistance gene. We used BL21(DE3) E. coli for the production of our proteins, and amplified some of the plasmid DNA in DH5α E. coli for future experiments. Heat shock transformation was used for all transformations. For the transformations, we used competent DH5α and BL21 cells from our lab and also prepared competent BL21(DE3) cells ourselves after we didn’t get any colonies at first (Fig. Fig 1 and Fig 2 ). We then validated the transformed clones by sending in colonies of BL21(DE3) for sequencing. To cover the complete insert, we chose standard primers for the T7 promoter and the T7 terminator. Both our constructs were synthesized correctly and showed no changes in sequence (results not shown).

Figure 1
Figure 1: DH5α E. coli transformed with aGFP_his (5 ng of plasmid DNA added to 50 μL of competent cells). Cells were plated on LB agar plates with 50 μg/mL kanamycin and incubated overnight at 37 °C.
Figure 2
Figure 2: BL21(DE3) E. coli transformed with GFP_his (10 ng of plasmid DNA added to 100 μL of competent cells). Cells were plated on LB agar plates with 50 μg/mL kanamycin and incubated overnight at 37 °C.

Testing our first constructs using protein precipitation

We prepared 30 mL liquid cultures of the transformed BL21(DE3) colonies ( Fig 3 ) and induced them with 0.5 mM of IPTG. Protein production ran overnight at 18 °C and 160 rpm. As we expected our protein constructs to be secreted extracellularly, we pelleted the liquid cultures the next day, and processed cell pellets as well as the supernatant. To obtain higher protein concentrations in the supernatant, we precipitated any protein present using trichloracetic acid (TCA). We analyzed the protein production by SDS-PAGE and staining with Coomassie Blue. To be able to compare the naturally occurring proteins in BL21(DE3) E. coli with recombinant protein production, we did not induce one liquid culture for each protein construct, respectively. However, we were not able to see any bands indicating recombinant protein production at the expected sizes of 20 kDa for the nanobody construct ( Fig. 4 ). Both gels showed weak bands at 35 kDa in the supernatant of the induced samples, which would be the expected size for the GFP construct ( Fig. 5 ). However, the appearance in both nanobody and GFP samples indicated that these bands are not due to recombinant GFP production, but might be a naturally produced and secreted protein of E. coli.

Figure 3: Growth of liquid cultures of BL21(DE3) E. coli transformed with aGFP_his, GFP_his and positive control. An uninduced liquid culture of BL21(DE3) transformed with GFP_his served as a negative control. Culture volume = 30 mL. Incubation at 37 °C and 130 rpm. Induction of aGFP_his after 1.5 h, of GFP_his after 2.5 h and of PC after 3.5 h.
Figure 4
Figure 4: SDS-PAGE of aGFP_his. No band detectable at expected size of 20 kDa for nanobody construct. M = PageRuler™ unstained protein ladder (26614, Thermo Scientific). pre Ind. = pre-induction, post Ind. = post-induction. - = uninduced. + = induced with 0.5 mM IPTG. CP = cell pellet. SN = TCA-precipitated supernatant. Polyacrylamide concentration: 15%. Gel was run at 140 V for 1.25 h.
Figure 5
Figure 5: SDS-PAGE of GFP_his. A band at 35 kDa appears post induction in both induced and uninduced supernatant samples. M = PageRuler™ unstained protein ladder (26614, Thermo Scientific). pre Ind. = pre-induction, post Ind. = post-induction. - = uninduced. + = induced with 0.5 mM IPTG. CP = cell pellet. SN = TCA-precipitated supernatant. Polyacrylamide concentration: 12%. Gel was run at 140 V for 1.25 h.

Testing our first constructs using Ni-NTA purification

As our gels showed no visible bands of recombinant proteins in the supernatant, we used Ni-NTA purification to concentrate and isolate our proteins. We first experimented with Ni-NTA resin and purification by centrifugation, but saw no definite results on the SDS-PAGE after staining (results not shown). We repeated the experiment using Ni-NTA resin in a gravity flow setup to be able to process bigger volumes of supernatant from 300 mL liquid cultures. Additionally, we introduced a negative control and a positive control for our protein production. The negative control consisted of an uninduced liquid culture, while our positive control was a his-tagged protein from the lab which was also under the control of the lac-Operon and a T7 promotor. This positive control, a phospholipase C (PLCN), was not expected to be secreted extracellularly, but was produced in the cytosol of the E. coli. Protein production after induction with IPTG ran for 3 h at 37 °C and 130 rpm.

We were able to detect the positive control in the cell pellet, but did not find any bands at the expected sizes again for the nanobody ( Fig. 6 ) or GFP constructs (results not shown).

Figure 6
Figure 6: SDS-PAGE of aGFP_his. No band detectable at expected size of 20 kDa for nanobody construct. Positive control shows a band at expected size of 79 kDa. M = PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (26616, Thermo Scientific). - = uninduced GFP_his (negative control). + = PLCN (positive control). CP = cell pellet. FT = flowthrough. W1 & W2 = wash steps 1 and 2. E1-4 = elution steps 1-4. Polyacrylamide concentration: 15%. Gel was run at 140 V for 1.25 h.

Testing our redesigned and modified constructs using protein precipitation

We reviewed our construct design and ordered two new construct variations: in one we decreased the distance between RBS and CDS from seven to one basepair (RBS1_GFP_his and RBS1_aGFP_his). For the second variation, we switched out the RBS derived from an iGEM part with the RBS commonly used in pET vectors (RBSp_GFP_his and RBSp_aGFP_his). Additionally, we used restriction digestion to remove the 3x NLS from our original two constructs, ligated the plasmids again and called the new constructs GFP_woNLS and aGFP_woNLS (results not shown). We verified the result of the restriction digestion and ligation by sequencing of the respective colonies using standard primers for T7 promoter and terminator again, and saw correct results with no changes in sequence ( Fig. 7 ).

Figure 7
Figure 7: Sequencing results for aGFP_woNLS_his clone 1 Original sequence with annotations shown at the top, sequencing result with trace data shown at the bottom. Sequence in the area around the recognition site of the EcoRI restriction enzyme is correct.

While waiting on RBSp-plasmids, we tried to express our RBS1-constructs and the constructs without NLS in 100 mL liquid cultures. We switched to a higher concentration of 1 mM IPTG for the induction and incubated overnight at 25 °C and 160 rpm. To quickly check if we obtained any secreted protein in the supernatant, we performed protein precipitation using TCA again. To open up the cells in the pellet and potential inclusion bodies, we sonified the cell pellets. After analysis with SDS-PAGE and Coomassie Blue staining, we detected a band at 70 kDa in the negative control and none in the positive control, which suggests that both samples had been switched. We found strong bands above 35 kDa again in the precipitated supernatants of RBS1_aGFP_his samples ( Fig. 8 ), but none in the samples of the constructs without NLS ( Fig. 9 ). Due to issues during gel preparation and damaged wells, samples were flowing out of wells into adjacent ones, which further impacted the analysis of the results.

Figure 8
Figure 8: SDS-PAGE of RBS1_aGFP_his. No band detectable at expected size of 20 kDa for nanobody construct. Negative control shows a band at size of 70 kDa, possibly switched with positive control. Strong bands appear above 35 kDa in precipitated supernatant samples. M = PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (26616, Thermo Scientific). - = uninduced GFP_his (negative control). + = PLCN (positive control). CP = cell pellet. SN = Supernatant. 1-3 = RBS1_aGFP_his clones 1-3. Polyacrylamide concentration: 15%. Gel was run at 140 V for 1.25 h.
Figure 9
Figure 9: SDS-PAGE of RBS1_GFP_his and constructs without NLS. No band detectable at expected size of 20 kDa for nanobody construct. Negative control shows a band at size of 70 kDa, possibly switched with positive control. RBS1_GFP_his supernatant shows a strong band above 35 kDa, similar to supernatant samples of RBS1_aGFP_his supernatant. M = PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (26616, Thermo Scientific). - = uninduced GFP_his (negative control). + = PLCN (positive control). CP = sonified cell pellet. SN = Supernatant. GFP = RBS1_GFP_his. 1 and 2 = GFP_woNLS_his clones 1 and 2. 3 and 4 = aGFP_woNLS_his clones 3 and 4. Polyacrylamide concentration: 12%. Gel was run at 140 V for 1.25 h.

Analyzing the mystery band at 35 kDa

We decided to repeat the analysis of the RBS1-constructs and the newly arrived RBSp-constructs to verify the strong bands above 35 kDa. This time, we also precipitated supernatants of the negative and positive controls in case they’d also show the 35 kDa band. Additionally, we decided to perform Ni-NTA purification again to validate if those bands could be recombinantly produced protein carrying a his-tag. We prepared 200 mL liquid cultures Fig. 10 and incubated them overnight with 1 mM IPTG at 25 °C and 150 rpm.

Figure 10: Growth of liquid cultures of BL21(DE3). E. coli transformed with aGFP_his, GFP_his and positive control. An uninduced liquid culture of BL21(DE3) transformed with GFP_his served as a negative control. Culture volume = 30 mL. Incubation at 37 °C and 130 rpm. Induction of aGFP_his after 1.5 h, of GFP_his after 2.5 h and of PC after 3.5 h.

Analysis by SDS-PAGE and Coomassie Blue staining showed the band above 35 kDa for each precipitated supernatant sample of each construct, and additionally weaker bands at this size in the positive control supernatant. Samples from the Ni-NTA purification showed no bands at all (results not shown). We did research on native proteins that are secreted in BL21(DE3) E. coli and found that outer membrane proteins like OmpA or OmpF can be released as well1. These two proteins, with molecular weights of 37 kDa and 39 kDa, respectively, would match the band at 35 kDa that we saw repeatedly. After reaching out to Dr. Schäfer, regarding his experience with nanobodies, he explained that the observed band might also represent the lacI protein with an approximate molecular weight of 38.6 kDa. This protein is part of the lac-Operon present in BL21(DE3) E. coli as well as on our introduced plasmid and might be produced in greater amounts than other native proteins.

Finally finding our proteins for the first time using Western Blot

We repeated the SDS-PAGE with the cell pellet and precipitated supernatant samples and performed a Western Blot. We used a monoclonal mouse antibody against 6x his-tags conjugated to horseradish peroxidase for visualization of the bands. As we forgot to dilute the positive control, the signal of the band was far too strong. However, we found bands corresponding to the respective construct sizes in cell pellet samples of RBSp_GFP_his and RBSp_aGFP_his. Additionally, we found two bands at 35 kDa and 20 kDa in the cell pellet samples of RBS1_GFP_his ( Fig. 11 ).

Figure 11
Figure 11: Western Blot of RBS1- and RBSp-constructs. Positive control shows a strong band at size of 70 kDa in the cell pellet. RBS1_GFP_his cell pellet shows bands at 35 and 20 kDa. Cell pellet samples of RBSp_GFP_his and RBSp_aGFP_his show weak bands at 35 kDa and 20 kDa, respectively. M = PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (26616, Thermo Scientific). + = PLCN (positive control). CP = sonified cell pellet. SN = Supernatant. 1 = RBS1_GFP_his. 2 = RBS1_aGFP_his. 3 = RBSp_GFP_his. 4 = RBSp_aGFP_his. Antibody: Anti-6X His tag® antibody [AD1.1.10] - HRP conjugated (ab178563, abcam). Antibody concentration: 1:2000. Polyacrylamide concentration: 12%. Gel was run at 140 V for 1 h. Imaging was performed using the ChemiDoc MP Imaging System (BIO-RAD). Exposure time: 25 s.

Unfortunately losing our proteins again

In our final week in the lab, we wanted to repeat the expression of the RBSp-constructs. Additionally, we transformed BL21(DE3) E. coli with our newly arrived constructs which designed in a way compatible with the iGEM Parts Registry (part_GFP_his and part_aGFP_his) and lacking protease sites and the linker responsible for extracellular secretion (part_bas_GFP_his and part_bas_aGFP_his). However, the competent BL21(DE3) cells we used seemed to have lost competency, as we only obtained two colonies for cells transformed with part_aGFP_his and one colony for those transformed with part_bas_aGFP_his. We still decided to use those clones, RBSp_aGFP_his-transformed BL21(DE3) cells and BL21(DE3) cells transformed with the positive control (the last two from cryostocks) to prepare overnight cultures for downstream preparation of 200 mL liquid cultures. Unfortunately, the overnight cultures for the positive control and the RBSp_aGFP_his-transformed cells did not grow. We prepared 200 mL liquid cultures for those using cells directly from the cryostock, but could not detect any growth after 4 h. Nevertheless, we proceeded with cultures of part_aGFP_his- and part_bas_aGFP_his-transformed BL21(DE3) cells which we incubated overnight at 25 °C with 1 mM IPTG for protein production. We decided to use 100 mL for sonification of the cell pellet as usual, but perform a periplasmic extraction through osmotic shock on the other 100 mL of the cultures, respectively. This way, we wanted to analyse if the protein constructs were transported into the periplasm of the cells. We analyzed both sonified cell pellets, the cytosolic fraction of the periplasmic extraction and the supernatant containing periplasmic proteins by SDS-PAGE and a subsequent Western Blot. Unfortunately, we could not detect any bands on the Western Blot ( Fig. 12 ). Since we did not have a positive control for this experiment, it is not possible to analyze potential errors. There could have been degradation issues with the antibody used for detecting the his-tagged proteins, or the dilution of the respective proteins might have been too high. Additionally, it might be that the new constructs were simply not expressed due to different reasons like issues during translation initiation at the RBS, translation disruption or folding issues with subsequent degradation. This is unlikely though, as the construct part_aGFP_his only differs from RBSp_aGFP_his in terms of restriction enzyme sites and a decreased distance by six base pairs between lac operon and RBS in the region upstream of the coding sequence. However, we cannot rule out any issues arising from these differences.

Figure 12
Figure 12: Western Blot of part_aGFP_his and part_bas_aGFP_his. Sonified cell pellets, cytosolic and periplasmic fractions were analyzed for each construct. No bands were detected. M = PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (26616, Thermo Scientific). CP = sonified cell pellet. C = cytosolic fraction after periplasmic extraction. PP = periplasmic fraction after periplasmic extraction. Antibody: Anti-6X His tag® antibody [AD1.1.10] - HRP conjugated (ab178563, abcam). Antibody concentration: 1:2000. Polyacrylamide concentration: 12%. Gel was run at 140 V for 1 h. Imaging was performed using the ChemiDoc MP Imaging System (BIO-RAD). Exposure time: 40 s.

Conclusion

As we as we had to finish our WetLab efforts due to wiki freeze, we could not continue to analyze the protein production. We conclude that our constructs for the anti-GFP nanobody and GFP are expressed in BL21(DE3) E. coli. However, we suspect that the expression is not optimal, as only small amounts of protein could be detected in a Western Blot. More thorough optimization with detailed analyses of expression conditions such as temperature, duration of induction with IPTG and IPTG concentration would be needed to improve protein production efficiency. Additionally, different kinds of extracellular secretion signals should be tested to facilitate secretion of the constructs into the medium.

Lipid Nanoparticles

Liposomes are a versatile and biocompatible platform for protein delivery. Given their suitability for therapeutic applications, we began the design of our liposome constructs by collecting key requirements from both therapeutic and production perspectives.

As ɑ-amanitin predominantly accumulates in hepatocytes, the delivery system should specifically target those cells to enable a sufficient therapeutic effect. Liposomes naturally acquire a protein corona upon administration in the blood, primarily composed of Apolipoprotein E (ApoE). This corona facilitates hepatocyte uptake via LDL receptor–mediated endocytosis. [13] [14]

The primary goal is the targeted delivery of the nanobody to hepatocytes for an improved therapeutic effect. Encapsulation within liposomes protects the nanobody from degradation and environmental factors until endocytosis took place and ensures its delivery to target cells at the same time. [8] [9]

Following endocytosis of nanobody-loaded liposomes into hepatocytes, they need to release their cargo into the cell before getting cleared. This process is called endosomal escape and there are several theories how it can be achieved. One of the most prominent is the proton sponge effect where ionizable lipids buffer the acidification in an endosome. This leads to further influx of protons and also chloride ions which increases the osmotic pressure. Eventually, the endosome will burst and the cargo is released into the cell. Another possibility is the fusion of endosomal and liposomal membranes also leading to a cargo release. [17]

With the help of certain modifications such as incorporating PEG-lipids (lipid molecules to which polyethylene is attached) into the liposome membrane, a good long-term stability can be achieved and storage up to six months is possible. [16]

For clinical translation, liposomes must be producible at large scale. Existing knowledge from lipid nanoparticle (LNP) production for mRNA vaccines provides valuable insights that can be adapted for protein delivery.

Design

For our project, we decided to produce liposomes made of three main components: soy phosphatidylcholine (PC), dioleoylphosphatidylethanolamine (DOPE) and cholesterol. Each component was selected based on its biophysical properties, biological compatibility, and functional contribution to the delivery process. The soy lipid as a natural lipid leads to good biocompatibility and low immunogenicity. Furthermore, it is known to form stable lipid bilayers which ensures the protection of the encapsulated nanobody. DOPE is added to facilitate the release of the cargo once the liposome is endocytosed. In the acidic environment of endosomes, DOPE tends to adopt a non-bilayer inverted hexagonal phase, which promotes the fusion of liposomal and endosomal membranes. The combination of these two lipids creates a liposome suitable for therapeutic application. Cholesterol as the third component improves the membrane stability and integrity of the liposome during circulation and also prevents leakage of the nanobody before reaching the target cells. Soy PC and DOPE additionally show the inherent ability to target hepatocytes. When administered into the blood, a protein corona of mainly Apolipoprotein E (ApoE) will be formed on the liposomes surface. This corona mediates endocytosis through low-density lipoprotein receptors on hepatocytes and thus enables the targeted delivery of the nanobody. [8] [9] [13] [14]

To test our initial fabrication pipeline, we prepared a liposome solution of PC and Cholesterol via thin -film-layer hydration. The thin-film-layer hydration technique allows reproducible vesicle formation. Later, the formed vesicles can be further processed by extrusion to optimize the size distribution.
Due to time constraints, we were not able to perform characterization and encapsulation experiments. Nevertheless, a good starting point for future investigations was created.

Theoretical Workflow for Lipid Nanoparticle (LNP) Characterization

The iGEM team collaborated with a partner laboratory at the Centre for Structural Systems Biology (CSSB), the Grünewald group, to design a workflow for lipid nanoparticle (LNP) characterization and validation. Due to limited time, the experiments were only partly performed, but the theoretical workflow outlines a feasible strategy for future implementation.

Figure 1
Figure 1: collection of the lipids available and used in collaboration with the Grünewald-Lab

LNP Generation

The workflow begins with the production of lipid films via freeze-thaw cycles, allowing lipids to self-assemble into nanoparticles. After formation, the LNPs are divided into aliquots for downstream processing, including extrusion and analytical characterization.

Proposed Refinements for Optimization

  • Pre-labeling of Lipids Before Rehydration
    • Suitable lipophilic dyes include:
      • Rhodamine-DHPE, DiI, DiD
      • Lipid-conjugated Alexa Fluor dyes (e.g., Alexa 647 C18)
  • Freeze-Thaw Processing After Rehydration

    Freeze-thaw cycles promote swelling of the lipid film and the formation of micellar structures. Vigorous agitation during freezing enhances micelle formation, which can be used to co-encapsulate proteins.

    Figure 2
    Figure 2: Laboratory equipment used for LNP preparation and the Freeze-Thaw Process

    Example: A recombinant nanobody can be added to the buffer and dye solution during freeze-thaw cycles, resulting in hybrid LNPs containing both nanobody and fluorescent lipid.

  • Extrusion for Size Homogeneity

    Following formation and encapsulation, liposomes are extruded to generate a uniform particle size, targeting diameters below 50 nm. This size range allows for multiple nanobodies encapsulated per particle. Size homogeneity is an important feature of LNPs used for therapeutic applications, provides reproducibility and allows a homogenous packaging of nanobodies.

  • Preparation for Characterization

    Extruded LNPs are carefully transferred to fresh tubes and subjected to analytical techniques to assess size, structure, and cargo incorporation. Proposed methods include:

    Method Advantages Limitations
    Dynamic Light Scattering (DLS) Fast particle size analysis , low sample volume Poor resolution for polydisperse samples
    Transmission Electron Microscopy (TEM) Direct structural visualization Labor-intensive, low statistical sampling
    Nanoparticle Tracking Analysis (NTA) Single-particle resolution for size distribution Requires specialized equipment and software
    Charge Detection Mass Spectrometry (CDMS) Measures particle mass and packaging Not yet standard in routine LNP workflows
    Fluorescence Labeling Enables imaging, tracking, and colocalization Incorporation efficiency may vary

While these procedures were not implemented during the iGEM project due to time constraints, the proposed workflow provides a conceptual blueprint for future LNP development. It integrates strategies for visualization, uniform sizing, nanobody encapsulation, and detailed characterization to ensure functional delivery to target cells.

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